Project Summary This project brings together structural, biochemical, proteomic and genetic expertise at UCSF to provide a detailed understanding of the structure and post-translational regulation of a key virulence factor in Mycobacterium tuberculosis. A hallmark of virulent, intracellular bacteria is their use of specialized secretion systems to inject proteins into mammalian cells. Multiple such systems are known in Gram-negative bacteria (termed the Type I-VI and VIII-IX) systems), but only one is found exclusively in Gram-positive bacteria: the type VII secretion (T7S) system. Mycobacterium tuberculosis uses multiple T7S systems to promote infection through secretion of effectors. In fact, the main genetic difference between the harmless vaccine strain BCG and pathogenic M. tuberculosis is the loss of the ESX-1 T7S system. T7S is also essential for abscess formation in Staphylococcus aureus and other pathogens may require T7S for virulence, as these systems are broadly conserved in Gram-positive bacteria. In this proposal we aim to build on our preliminary data regarding the basic mechanisms of T7S and unravel the biochemical and structural foundations of this complex biological machine.